The present invention relates to a component analyzing apparatus with a microchip suitable for analysis in the field of, mainly, biochemistry, molecular biology and clinical medicine, and especially for analyzing DNA and protein.
A capillary electrophoresis (CE) is a method suitable for separating components having similar structures, such as optical separation and separation of isomers, as well as analyzing bio-components such as peptides, proteins, DNA and sugars. The capillary electrophoresis has been widely used for monitoring clinical medicine, drugs, environment materials or the like. Especially, a microchip type apparatus, i.e. a microchip electrophoresis apparatus, having a micro-flow path using a photo-lithographic technique or the like, is easy to handle and has been widely used for analyzing DNA or the like.
FIGS. 8(a)–8(c) show an example of an electrophoretic chip disclosed in Japanese Patents No. 3077609 and No. 3417344; and FIG. 9 is a perspective view thereof. An electrophoretic chip 10 is formed of a pair of transparent flat plates 11 and 15 made of glass, quartz or the like. A sample drawing groove 12 and a migration groove 13 crossing each other at a crossing portion (hereinafter, called a crossing portion 14) are formed in an upper surface of the lower transparent flat plate 11 (refer to FIG. 8(b)). Through-holes 16 are provided in the upper transparent flat plate 15 at positions corresponding to the respective end portions of the grooves 12 and 13 (refer to FIG. 8(a)). The grooves 12 and 13 are provided in the surface of the transparent flat plate 11, for example, by etching, and a width of the groove portions is about 10–100 μm and a depth thereof is about 5–50 μm.
As shown in FIG. 8(c), a pair of the transparent flat plates 11 and 15 is bonded together so that the grooves 12 and 13 are positioned inside the flat plates 11 and 15. Accordingly, a sample introducing flow path 17 communicating with outside through the first and second reservoirs R1 and R2 formed by the through-holes 16 and a separating flow path 18 communicating with outside through the third and fourth reservoirs R3 and R4 are formed in the electrophoretic chip 10. The respective reservoirs R1 to R4 receive capillaries therein for introducing a liquid, such as a migration liquid, from outside. Electrodes (not shown) for applying a voltage to a migration liquid reserved in the reservoirs are provided, respectively. The electrode is formed of, for example, a conductive thin film formed by photolithography on the surface of one of the transparent flat plates, and extends to the end surface of the transparent flat plate.
A general procedure of measurement using such an electrophoretic chip 10 is as follows. First, the whole flow paths are filled with the migration liquid. Then, the first reservoir R1 is filled with a small amount (about 1–2 μl) of the sample solution, and a high voltage where a flow design and a migration condition are optimized is applied to the respective reservoirs R1 to R4. As an example, a voltage of 0.6 kV is applied to the first and fourth reservoirs R1 and R4; a voltage of 0.3 kV is applied to the third reservoir R3; and the second reservoir R2 is grounded. Then, the sample injected into the first reservoir R1 flows into the sample introducing flow path 17 due to an electric potential difference, passes through the crossing portion 14 and moves toward the second reservoir R2. At this time, the migration liquid also moves toward the second reservoir R2 from the third and fourth reservoirs R3 and R4, respectively, so that the sample does not diffuse to the separating flow path 18 during the sample introducing period.
Then, a voltage of 0.2 kV is applied to the first and second reservoirs R1 and R2; a voltage of 0.5 kV is applied to the third reservoir R3; and the fourth reservoir R4 is grounded. Accordingly, the sample present at the crossing portion 14 is introduced into the separating flow path 18 due to an electric potential difference, and migrates toward the fourth reservoir R4. In the sample introducing flow path 17, the sample present on a side of the first reservoir R1 from the crossing portion 14 moves back to the first reservoir R1; and the sample present on a side of the second reservoir R2 from the crossing portion 14 moves back to the second reservoir R2. Therefore, only the sample in a predetermined quantity according to a volume of the crossing portion 14 is introduced into the separating flow path 18.
As described (above, when the sample migrates in the separating flow path 18, the respective components contained in the sample are separated. With respect to the sample separated as described above, an electroferrogram, where an abscissa axis represents a migration time, is obtained through one point UV absorption detection using an ultraviolet visible absorption detector or through one point fluorescence detection using a fluorescence detector having an incident-light fluorescence optical system.
When the sample is detected at just one point on the separating flow path 18, it is difficult to obtain a whole image of the sample components separating and moving along the separating flow path 18. In the UV absorption detection using the ultraviolet visible absorption detector as disclosed in Japanese Patent No. 3077609, it is possible to obtain an image of the sample components spreading in a predetermined area along the separating flow path 18. However, the sensitivity of the UV detection using light absorption of the sample itself is inferior to that of the fluorescent detection. Accordingly, it is difficult to detect a component having a low concentration or low light absorption. The fluorescent detection is not greatly affected by background light as compared with the UV detection. However, it is necessary to reduce the effect of the background light for further improving the accuracy and the sensitivity.
In view of the above problems, an object of the invention is to provide a component analyzing apparatus using a component analyzing microchip for separating sample components through an electrophoretic migration, a liquid-supply pump or the like, wherein the separated sample components are detected over a predetermined area along a flow path at a high sensitivity, not at one point on the separating flow path of the microchip.
Further objects and advantages of the invention will be apparent from the following description of the invention.